Hematopoiesis is the process of blood cell production which takes place in the bone marrow. Stem cells in the bone marrow are the progenitor cells for all of the various cell types found in the circulating blood. These stem cells are functionally defined by their capacity to repopulate, on a long-term basis, all of the hematopoietic cell lineages in a lethally irradiated animal [Nicola, N. A. (1993) in Application of Basic Science to Hematopoiesis and the Treatment of Disease, E. D. Thomas and S. K. Carter (ed), Raven Press, New York]. Through a complex series of regulatory events, stem cells differentiate into a number of types of cells including at least red blood cells, leukocytes, lymphocytes, platelets (thrombocytes), monocytes, macrophages, mast cells, basophils, eosinophils, .beta.-lymphocytes and T-lymphocytes. Millions of each type of new blood cells are produced daily and are released into the circulating blood to replace destroyed blood cells and maintain homeostasis. (Nathan, D. G. (1992) in Cecil Textbook of Medicine, L. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed., W. B. Saunders Co, Philadelphia, pages 817-836).
The production of the different cell types can be modulated in response to exogenous stimuli such as infection or blood loss. For example, during infection white blood cells (leukocytes) are mobilized from peripheral stores, e.g. along the margins of vascular walls (the so-called process of de-marginalization) and there is a concomitant increase of leukocyte production in the bone marrow (Bagby, G. C. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed., W. B. Saunders Co., Philadelphia, pages 914-920). Acute blood losses such as menstruation, trauma or surgical blood loss may result in anemia wherein the blood is deficient in red blood cells, in hemoglobin or in total volume (hematocrit&lt;40%, hemoglobin&lt;12 grams/dl, red blood cells&lt;4.times.10.sup.6 /ul, or mean cell volume&lt;80 fl; Nathan, D. G. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed., W. B. Saunders Co., Philadelphia, pages 817-836). The red cell mass (total red blood cells, either total number, weight or volume) acts as an organ that delivers oxygen to tissues. Red cell mass and the rate of red blood cell production are closely coupled to the supply and demand for oxygen in body tissues. Red blood cell production is stimulated by low tissue tension of oxygen. Anemic conditions result in reduced oxygen levels in tissues (hypoxia). Hypoxia in the kidney is sensed by the renal parenchyma which stimulates the release of erythropoietin from the kidney. Erythropoietin is the major regulatory hormone of erythropoiesis produced in response to hypoxia resulting from alterations in the red cell mass. (Erslev, A. J. (1990) in Hematology, W. J. Williams, E. Beutler, A. J. Erslev and M. A. Lichtman eds, McGraw-Hill, Inc. New York, pp 389-407).
Other deficits in specific circulating cell types may occur as well. Leukopenia, a general term that describes decreases in any one of a number of different leukocyte cell populations, may result from a de-coupling of the process of demargination and the rate of replacement of cells differentiated from progenitor bone marrow cell lines (Bagby, G. C. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed. W. B. Saunders Co., Philadelphia, pages 914-920). Neutropenia, a decrease in circulating neutrophils to &lt;2.times.10.sup.9 cells per liter, results in a greatly increased risk of severe bacterial infection (Kaplan, M. E. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed. W. B. Saunders Co, Philadelphia, pages 907-914). Thrombocytopenias are defined as decreases in circulating platelet levels to approximately &lt;100,000/.mu.L (Shuman, M. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed. W. B. Saunders Co, Philadelphia, pages 987-999). Low circulating thrombocytes may be the result of a number of underlying conditions such as bone marrow injury, the utilization of chemotoxic agents, suppression of the bone marrow due to chemotherapeutic or radiotherapeutic agents, heavy metal poisoning, hemolytic uremic syndrome, HIV infection, tuberculosis, aplastic anemia, thrombotic thrombocytopenic purpura, and immune disorders such as idiopathic thrombocytopenic purpura, leukemias, and myelofibrosis. These thrombocytopenias can result in life-threatening uncontrolled bleeding (Shuman, M. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed. W. B. Saunders Co, Philadelphia, pages 987-999).
Each of these disturbances in hematopoiesis may result in an upregulation of the bone marrow differentiation processes which re-supplies the deficient cell population. However, some disturbances in hematopoiesis are so severe that therapeutic intervention is required.
The broad range of cytopenias (decreases in the circulating population of any given blood cell type) can be treated with only a limited number of therapeutic modalities. For example, management of neutropenia is generally limited to treatment of the underlying disease state that results in neutropenia. Such diseases include Felty's syndrome, myelodysplasia, hypersplenism, some cancers, and bone marrow compromises resulting from, for example, toxic chemotherapeutic drugs or toxins. There are few treatments specifically designed to increase neutrophil levels in the blood. These treatments include:
(1) lithium carbonate treatment, although this has been found to have significant toxicity PA1 (2) immunosuppressive therapy, typically reserved for treatment of patients whose neutropenia is the result of an autoimmunologically mediated destruction of neutrophils PA1 (3) bone marrow transplantation, which, although effective if successful, is associated with significant mortality, and PA1 (4) neutrophil transfusion, which can be costly and is ineffective unless maintained for a significant period of time due to the very short half life of neutrophils in the blood stream (Kaplan, M. E. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed. W. B. Saunders Co, Philadelphia, pages 907-914).
Treatment for thrombocytopenia is typically an infusion of platelets. Platelet infusions, as with infusions from any human derived blood product, carry significant risk for transmission of infective agents. Moreover, repeated transfusions of platelets may cause the formation of multiple alloantibodies which result in not only the destruction of the transfused platelets but also the destruction of the patient's endogenous thrombocyte population.
Anemias can be resolved by treating the underlying cause of the anemia, such as renal failure, liver disease, endocrine disorders, parvovirus, Epstein-Barr virus, or hepatitis C virus. However, the direct formation of red blood cells can be stimulated with a limited number of therapeutics. Treatments include administration of iron, hemin (a source of iron) or erythropoietin, a naturally occurring or recombinantly produced hematopoietic growth factor. The efficacy of iron or hemin therapy is limited due to poor bio-availability of the iron in these compounds as well as toxicity of the high dosages required to enhance erythropoiesis. In addition, iron therapy is not useful for anemias that are not due to simple iron deficiency. Erythropoietin therapy is limited because it does not appear to be effective in mobilizing endogenous iron stores and only enhances production of erythroid progenitor cells. Without mobilization of these iron stores, erythropoiesis cannot be sustained. For example, regular administration of recombinant erythropoietin to dialysis patients with chronic renal failure-induced anemia results in sustained erythropoiesis and an increase in hematocrit. However, continued erythropoiesis in this situation frequently results in iron deficiency that can limit the long term effectiveness of this treatment modality (Grutzmacher, P. (1992) Clin. Nephrol., 38: S92-S97).
The most promising therapeutic modalities for treatment of a number of cytopenias center on the administration of hematopoietic growth factors. Neutropenia has been treated with administration of with GM-CSF or G-CSF (granulocyte-macrophage colony stimulating factor and granulocyte stimulating factor, respectively). As discussed above, anemia in chronic renal failure has been treated with erythropoietin.
However, the hematopoietic system is complex; sustained, enhanced hematopoiesis is a complex interaction between growth factors, inhibitors and receptors. To date, at least twenty growth factors have been recognized (Nicola, N. A. (1993) in Application of Basic Science to Hematopoiesis and the Treatment of Disease, E. D. Thomas and S. K. Carter (ed), Raven Press, New York). These include Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Macrophage Colony-Stimulating Factor (M-CSF), Granulocyte Colony-Stimulating Factor (G-CSF), Stem Cell Factor (SCF), Erythropoietin (EPO) and Interleukins 1-13 (IL1 to IL7) [Quesenberry, P. J. (1990) in Hematology, W. J. Williams, E. Beutler, A. J. Erslev and M. A. Lichtman (eds), McGraw-Hill, Inc. New York, pp 129-147; Nicola, N. A. (1993) in Application of Basic Science to Hematopoiesis and the Treatment of Disease, E. D. Thomas and S. K. Carter (ed), Raven Press, New York).
A number of these growth factors have been cloned and expressed recombinantly. These include G-CSF (Souza, L. M., U.S. Pat. No. 4,810,643), M-CSF (Clark, S. C. and Wong, G. G., U.S. Pat. No. 4,868,119), IL-3 (Biasdale, J. H. C., EP 355093), erythropoietin (Lin, U.S. Pat. No. 4,703,008), stem cell factor (Zsebo, K. M. et al., PCT/US90/05548), and GM-CSF (Deeley M., et al., U.S. Pat. No. 5,023,676). Administration of these alone or in combination have resulted in significant hematopoiesis both in vitro and in vivo (Mertelsmann, R. H. (1993) in Application of Basic Science to Hematopoiesis and the Treatment of Disease, E. D. Thomas and S. K. Carter (ed), Raven Press, New York; Williams, U.S. Pat. No. 5,032,396; Zsebo et al., PCT/US90/05548; Gillis, S. U.S. Pat. No. 5,199,942; Donahue, R. E., U.S. Pat. No. 5,198,417). Of these growth factors, erythropoietin is unusual in that it acts as a hematopoietic regulator which is selective for one cell lineage, the red blood cell lineage. Most hematopoietic growth factors are not specific and affect, to a different extent, multiple hematopoietic cell lines. Most hematopoietic growth factors are produced at multiple sites in the body, and many act locally and are rarely found in circulation (Nicola, N. A. (1993) in Application of Basic Science to Hematopoiesis and the Treatment of Disease, E. D. Thomas and S. K. Carter (ed), Raven Press, New York). Hematopoietic growth factors are present in extremely small amounts and are difficult to detect except in artificial culture systems and conditioned media. Moreover, hematopoietic growth factors act exclusively to modulate the growth and/or differentiation of the hematopoietic cell lines. The inventors of the present invention have surprisingly found that hemoglobin, whose major function is the transport of oxygen in the body and is found in high concentrations in red blood cells, when purified and administered in low dose, acts as a hematopoietic growth factor.
Early investigators had suggested that there was stimulation of hematopoiesis after administration of cell free hemoglobin (Hooper et al. (1920) Am. J. Physiol. 53: 263-282; Naswitis, K. (1922) Dtsch. med. Wochenschrift 48: 187-188; Furukawa, K. (1922) Klin. Wochenschrift 1: 723-725; Amberson (1937) Biol. Revs. 12: 48-86; Ferrari, R. (1932) Arch. Sci. Biologiche 27: 25-40; Hawkins and Johnson (1939) Am. J. Physiol. 126: 326-336). These investigators suggested that hemoglobin released from lysed red blood cells was the primary hematopoietic factor responsible for the enhancement of erythropoiesis either by direct action on hematopoietic tissues or by furnishing some material required for erythropoiesis, or by both actions (Amberson (1937) Biol. Revs. 12: 48-86 and references therein). Amberson et al. [(1949) J. Appl. Physiol. 1:469-489)] later observed an increase in reticulocyte count and hematocrit (both indicators of erythropoiesis) in 3 of 5 patients after administration of a crude hemoglobin solution. However, administration of the crude hemoglobin utilized in early studies resttlted in renal damage, anaphylaxis, and may have contributed to death (Amberson et al. [(1949) J. Appl. Physiol., 1: 469-489). These toxic effects of hemoglobin administration may have been due to contaminating elements such as stroma or endotoxins in the hemoglobin preparations or to the nature of the hemoglobin itself (DeVenuto et al. (1979) Surg. Gyn. Obstet. 149: 417-436; Sunder-Plassmarm et al. (1975) Europ. J. Intensive Care Med. 1: 3714 42; Feola, et al. (1990) Biomat. Art. Cell, Art. Org. 18:233-249). The untreated human hemoglobin tetraruer is composed of two .alpha. subunits and two .beta. subunits. The tetramer, if outside a red blood cell, will dissociate into two .alpha./.beta. dimers which can pass into the kidney and can cause renal failure at higher doses. Any uncrosslinked hemoglobin, when administered even in low doses is cleared from the body extremely rapidly, on the order of minutes. Stroma from poorly purified red blood cell preparations resulted in anaphylaxis. Thus, even though there was some evidence that hemoglobin administration might result in erythropoiesis, no hemoglobin solution existed which could be administered safely and effectively.
The enhancement of erythropoiesis upon the administration of crude hemoglobin solutions is consistent with the observation that anemia due to hemolysis is associated with a more pronounced erythroid hyperplasia and reticulocytosis than blood loss anemia of the same magnitude (Erslev, A. J. (1990) in Hematology, W. J. Williams, E. Beutler, A. J. Erslev and M. A. Lichtman (eds), McGraw-Hill, Inc. New York, pp 389-407).
In the late 1970's, work in multiple labs demonstrated that treatment of mouse erythroleukemia cells and normal bone marrow cells with hemin increased the synthesis of hemoglobin at the transcriptional level (Ross, J. and Sautner, D. (1976) Cell 8: 513; Dabhey, B. J. and Beaudet, A. L. (1977) Arch. Biochem. Biophys. 179: 106; Porter, P. N. et al. (1979) Exp. Hematol. 7: 11). Hemin is the chloride salt of oxidized heme whereas hemoglobin contains reduced heme in the heme pocket of each of the globin subunits. More recently, work by Monette and co-workers has shown that hemin acts synergistically with interleukin 3 to promote the growth of erythroid progenitor cells in vitro and in vivo (in mice). In a series of papers (Holden, S. A. et al. (1983) Exp. Hematol. 11: 953-960; Monette, F. C. et al., (1984) Exp. Hematol. 12: 782-787; Monette, F. C. and Sigounas, G. (1988) Exp. Hematol. 16: 727-729; Monette, F. C. (1989) Ann. New York Acad. Sci. 554: 49-58), Monette clearly demonstrated the capacity of hemin to augment directly and in a cell specific manner the proliferation and/or differentiation of primitive bone marrow erythroid progenitors. Kappas and Abraham have also observed the potentiation of erythroid progenitor cell growth with hemin administration in vitro (PCT publication PCT/US91/05283). However, none of these workers has examined or suggested the role of free hemoglobin in the stimulation of erythropoiesis, nor have any of these investigations suggested that hemoglobin itself may have an erythropoietic effect above and beyond the simple delivery of bioavailable iron.
Indeed, the erythropoietic effect of hemoglobin has been seen only when very large amounts of hemoglobin have been administered. For example, Feola et al. [(1992) Surg. Gyn. Obstet. 174: 379-386] administered high volumes of bovine hemoglobin solution (25% of the patient's total blood volume, approximately 17-35 grams of bovine hemoglobin, or a total of 1.75 g hemoglobin/kg of body weight) to sickle cell children in vaso-ocdusive or aplastic crisis. In this group of nine patients who ranged in age from 5-13 years of age, peripheral reticulocytes increased from 3.7.+-.3.9% to 49.+-.6.5% after 3 days and blood hemoglobin increased from 6.34.+-.2.0 gm/dl to 10.6.+-.1.3 gm/dl after 1 week. However, Feola et al. administered antibiotics and antimalarials concurrently with the hemoglobin treatment, and the mediation of the underlying infections alone may have resulted in erythropoiesis. Moreover, administration of such large amounts of any hemoglobin, particularly a nonhuman derived bovine hemoglobin, may result in unexpected and undesirable immunological effects. Feola et al. state that further examinations are required to determine whether immunologic reactions would develop upon repeated administration of a bovine-derived hemoglobin solution. High dose administration of hemoglobin can serve as simply a source of bioavailable iron, and is thus not significantly different from simple iron treatment of anemia. The inventors of the present invention have surprisingly found that low dose administration of a recombinant hemoglobin results in hematopoiesis as well.